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Application of Triazamidine in the Preparation of Furin Inhibitors

A technology of triazamidine and preparations, applied in the field of medicine

Active Publication Date: 2021-12-28
SHENYANG PHARMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the prior art, there is no relevant report on the use of triazine as a human furin inhibitor for the preparation of antiviral drugs

Method used

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  • Application of Triazamidine in the Preparation of Furin Inhibitors
  • Application of Triazamidine in the Preparation of Furin Inhibitors
  • Application of Triazamidine in the Preparation of Furin Inhibitors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Enzyme Activity Evaluation System Determines Triazamidine Targeted Inhibition of Furin Activity

[0026] 1. Protein Expression and Purification of Furin

[0027] The detailed steps of expression and purification of human furin (UNIPROT ID P09958) refer to previously reported work (Biochemistry, 2018, 57, 925-934), with some modifications. The gene sequence encoding amino acids 23-574 of human furin was cloned into a PEGMan vector with an N-terminal secretion signal peptide and a C-terminal His tag. The expression plasmid was transfected into HEK293 GnTI cells with polyethylenimine transfection reagent, and the transfected cells were cultured at 10 cm in 10 mL DMEM (Invitrogen) medium containing 10% fetal bovine serum (HyClone, GE Healthcare) Incubate overnight in the dish. Then, the medium was changed to 10 mL freeStyle 293 expression medium (Thermo Fisher Scientific), and the cells were cultured for 72 h. The medium supernatant was collected by centrifugat...

Embodiment 2

[0030] Example 2 Determination of the binding affinity between triazamidine and furin

[0031] 1. Furin expression and purification

[0032] The method is the same as in Example 1 Furin protein expression and purification

[0033] 2. Determination of binding affinity between triazine and furin by microthermophoresis

[0034] Micro-thermophoresis (MST) is a technique based on fluorescence detection and thermophoresis for detecting protein-protein or protein-small molecule interactions. To further validate the results of the enzyme activity assay, the binding affinity between triazamidine and furin was evaluated using the MST method. The result is as figure 2 Shown, analysis obtains the binding dissociation constant of triazamidine and furin to be (K d ) was 14.90±6.29 μM, indicating that triazine has a strong binding affinity with furin.

Embodiment 3

[0035] Example 3 Triazamidine Anti-Influenza A H1N1 Influenza Virus Activity Experiment

[0036] 1. Determination of virus titer

[0037] MDCK cells were seeded in a 96-well culture plate and cultured to a monolayer for experiments. The culture solution containing 10% serum in the original cell culture plate was removed, and the virus was -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 and 10-8 The dilution of the above-mentioned solution was added to the cell plate, and after adsorption at 35°C for 30 minutes, the cell maintenance solution was added, and a normal cell control was set up at the same time, and cultured at 35°C. Observe the lesions of the cells every day and record the results. Calculate TCID 50 =10 -4 / 100μL is 100.

[0038] 2. Determination of sample cytotoxicity

[0039] Select the virus-susceptible cell MDCK, add different concentrations of samples (μg / mL), culture for 7 days, observe the cytotoxicity every day, use the R-M formula to calculate the ...

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Abstract

The invention belongs to the technical field of medicine and relates to the application of diminazene in the preparation of antiviral drugs, in particular to the application of diminazene as a human furin inhibitor in the preparation of antiviral drugs. The virus is a virus carrying a furin cleavage site, and the virus includes: certain coronaviruses (such as novel coronaviruses), influenza viruses (such as H7N1, H5N1), retroviruses (such as HIV), nipple Oncoviruses (such as HPV), herpesviruses (such as human cytomegalovirus), flaviviruses (such as Zika virus), filoviruses (such as Ebola), etc.

Description

technical field [0001] The invention belongs to the technical field of medicine and relates to the application of diminazene in the preparation of antiviral drugs, in particular to the application of diminazene as a human furin inhibitor in the preparation of antiviral drugs. Background technique [0002] S protein (Spike glycoprotein) is a key protein for coronaviruses to bind to host receptors and enter cells. It plays a decisive role in the infectivity of coronaviruses. It consists of two parts: S1 that binds to host receptors and S2 that forms a membrane fusion protein machine Composition (Proc Natl Acad Sci USA, 2009, 106, 5871-5876). The enzymatic cleavage of S protein S1 is a necessary condition for exposing S2 to cause membrane fusion so that the virus can infect human cells. At the same time, enzyme cleavage at the S1 / S2 site can improve the flexibility of the S1 part of the virus and promote its adaptation to human receptors. Increased affinity (J Virol, 2011, 85,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/655A61P31/14A61P31/22A61P31/16
CPCA61K31/655A61P31/14A61P31/22A61P31/16Y02A50/30
Inventor 李华陈丽霞吴灿荣孙德娟刘洋杨月影
Owner SHENYANG PHARMA UNIVERSITY
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